Methods for extracting keratin proteins

ABSTRACT

Described herein are methods to produce keratin protein-based biomaterials, the parameters required to achieve improved extraction, the parameters required to improve isolation, the parameters of lyophilization and the grinding process to achieve consistent particulate sizes of protein materials.

1. FIELD OF THE INVENTION

This invention relates to methods to extract and purify keratinprotein-based biomaterials.

2. BACKGROUND OF THE INVENTION

Keratins are a family of proteins found in the hair, skin, and othertissues of vertebrates. Hair is a unique source of human keratinsbecause it is one of the few human tissues that are readily availableand inexpensive. Although other sources of keratins are acceptablefeedstocks for the present invention (e.g. wool, fur, horns, hooves,beaks, feathers, scales, and the like), human hair is preferred becauseof its biocompatibility in human medical applications.

Keratins can be extracted from human hair fibers by oxidation orreduction using methods that have been widely published in the art. Ifone employs a reductive treatment, the resulting keratins are referredto as kerateines. If an oxidative treatment is used, the resultingkeratins are referred to as keratoses. These methods typically employ atwo-step process whereby the crosslinked structure of keratins is brokendown by either oxidation or reduction. In these reactions, the disulfidebonds in cystine amino acid residues are cleaved, rendering the keratinssoluble without appreciable disruption of amide bonds. Many of thekeratins can remain trapped within the cuticle's protective structure,so a second-step using a denaturing solution is typically employed toeffect efficient extraction of the cortical proteins (alternatively, inthe case of oxidation reactions, these steps can be combined). This stephas also been widely published in the art as solutions such as urea,transition metal hydroxides, surfactant solutions, and combinationsthereof have been employed. Common methods include the use of aqueoussolutions of tris(hydroxymethyl) aminomethane in concentrations between0.1 and 1.0M, and urea solutions between 0.1 and 10M.

Many protein purification techniques are known in the art and range fromthe most simplistic such as fractional precipitation, to the mostcomplex such as immunoaffinity chromatography. For example, sub-familiesof acidic and basic keratins have been described as being separable bymoving bounding electrophoresis, but these fractions or their propertieshave not been described.

The methods that have been described in the art to extract theseproteins rely on a chemical process of oxidation or reduction with lessthan optimal extraction. Accordingly, there is a great need to providean optimized protein extraction procedure that provides a highly purekeratin protein product that retains structure and function.

3. SUMMARY OF THE INVENTION

Disclosed herein are methods to extract and purify keratin protein-basedbiomaterials. In some embodiments, the invention provides methods toproduce keratin protein-based biomaterials, the parameters required toachieve improved extraction, the parameters required to improveisolation, the parameters of lyophilization and the grinding process toachieve consistent particulate sizes of protein materials.

The invention also provides methods to extract keratin proteinscomprising: a) treating a keratin protein source with an oxidizing orreducing agent to solubilize keratin proteins; b) separating the solubleproteins from the keratin protein source by high speed centrifugation toproduce a clarified soluble keratin protein solution; and c)lyophilizing the clarified soluble keratin protein solution into akeratin protein cake, wherein degradation of said keratin protein isminimized.

The invention also provides methods to extract keratin proteinscomprising: a) treating a keratin protein source with an oxidizing orreducing agent to solubilize keratin proteins; b) separating the solubleproteins from the keratin protein source by high speed centrifugation toproduce a clarified soluble keratin protein solution; and c) dialyzingthe clarified soluble keratin protein solution wherein degradation ofsaid keratin protein is minimized.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a cartoon showing a potential mixing container thatprovides mechanical agitation

FIG. 2 is a scanning electron micrograph of hair shafts after 12 hoursof mechanical mixing in the presence of an oxidant.

FIG. 3 depicts hair after base extraction. FIG. 3A is a picture of hairafter base extraction. FIG. 3B is a scanning electron micrograph of ahair shaft after base extraction. The shaft is split in half and themajority of its cortical proteins (alpha and gamma keratins) have beenremoved during extraction, leaving the outer beta keratin hair covering.

FIG. 4 is a scanning electron micrograph of hair shafts after finalwater extraction. The majority of cortical proteins are extracted andonly the residual beta keratin hard coverings of the hair shafts appearto remain.

5. DETAILED DESCRIPTION

“Keratin protein source” as used herein includes proteinaceous sourcesof keratin proteins including but not limited human or animal wool, fur,horns, hooves, beaks, feathers, scales, and the like.

“Keratin protein(s)” as used herein collectively refers to keratin inkeratin protein sources, including but not limited to naturallyoccurring keratin, reduced keratin, and/or oxidized keratin, orS-sulfonated keratin. This term also refers to the extracted keratinderivatives that are produced by oxidative and/or reductive treatment ofkeratin, including but not limited to keratose, alpha-keratose,gamma-keratose, kerateine, alpha-kerateine, or gamma-kerateine.

“Clarified keratin protein solution” as used herein refers to a solutioncomprising extracted keratin proteins that has undergone at least onehigh speed centrifugation step to clarify the solution of othercontaminants.

“Keratin protein cake”, “lyophilized protein cake”, or “protein cake” asused herein includes but is not limited to a freeze-dried and/or vacuumdried keratin protein composition that exhibits long-term stability,short reconstitution time, uniform appearance, and low moisture content.Further, in some embodiments, “keratin protein cake”, “lyophilizedprotein cake”, or “protein cake” refers to compositions substantiallyfree of bulking agents or stabilizers.

Keratin Protein Sources

Keratins are a family of proteins found in the hair, skin, and othertissues of vertebrates. Hair is a common source of human keratinsbecause it is one of the few human tissues that is readily available andinexpensive. Other sources of keratins are acceptable feedstocks for thepresent invention, (e.g., wool, fur, horns, hooves, beaks, feathers,scales, and the like). Human hair is often used with human subjectsbecause of its biocompatibility. Accordingly, in some embodiments, humanhair is the keratin protein source The human hair can be end-cut, as onewould typically find in a barber shop or salon.

Keratin Proteins

Soluble keratins can be extracted from human hair fibers by oxidation orreduction using methods known in the art. These methods typically employa two-step process whereby the crosslinked structure of keratins isbroken down by either oxidation or reduction. In these reactions, thedisulfide bonds in cystine amino acid residues are cleaved, renderingthe keratins soluble. The cuticle is essentially unaffected by thistreatment, so the majority of the keratins remain trapped within thecuticle's protective structure. In order to extract these keratins, asecond step using a denaturing solution is employed. Alternatively, inthe case of reduction reactions, these steps can be combined. Denaturingsolutions known in the art include urea, transition metal hydroxides,surfactant solutions, and combinations thereof. Common methods useaqueous solutions of tris base(2-Amino-2-(hydroxymethyl)-1,3-propanediol) in concentrations between0.1 and 1.0 M, and urea solutions between 0.1 and 10M, for oxidation andreduction reactions, respectively.

If one employs an oxidative treatment, the resulting keratins arereferred to as “keratoses.” If a reductive treatment is used, theresulting keratins are referred to as “kerateines.”

Crude (unfractionated) extracts of keratins, regardless of redox state,can be further refined into matrix (KAP and gamma), alpha, and/orcharged (acidic or basic) fractions by a variety of methods such asisoelectric precipitation, dialysis, or high performance liquidchromatography (HPLC), as desired. In a crude extract, the alphafraction begins to precipitate below pH 6 and is essentially completelyprecipitated by pH 4.2.

In some embodiments, KAP co-precipitate with the alpha fraction, therebyproducing an alpha/KAP mixture.

High molecular weight keratins, or “alpha keratins,” (alpha helical),are thought to originate from the microfibrillar regions of the hairfollicle, and typically range in molecular weight from about 40-50kiloDaltons for monomers and 80-100 kiloDaltons for dimers. Lowmolecular weight keratins, or “gamma keratins,” or keratin-associatedproteins (globular), are thought to originate from the matrix regions ofthe hair follicle, and typically range in molecular weight from about3-30 kiloDaltons for KAP and 10-15 kiloDaltons for gamma keratins

In some embodiments, the keratin preparations (particularly alpha and/orgamma kerateine and alpha and/or gamma-keratose) have an averagemolecular weight of from about 10 to about 70 or about 85 or about 100kiloDaltons. Other keratin derivatives, particularly meta-keratins, mayhave higher average molecular weights, e.g., up to 200 or 300kiloDaltons.

Even though alpha and gamma keratins possess unique properties, theproperties of subfamilies of both alpha and gamma keratins can only berevealed through more sophisticated means of purification and separationsuch as provided herein. Additional properties that are beneficialemerge and can be optimized upon further separation and purification ofcrude keratin extracts.

Keratose Production

One method for the production of keratoses is by oxidation of keratinwith hydrogen peroxide, peracetic acid, or performic acid. In a specificembodiment, the oxidant is peracetic acid. Generally, a solution ofperacetic acid is used at a concentration range of about 1% to about10%. A specific concentration used can be a 2% solution of peraceticacid. In some embodiments, the oxidant concentrations range from a ratioof about 5:1 to about 50:1 weight to weight to the keratin proteinsource to be extracted. A specific embodiment uses a weight to weightratio of 25:1 of a 2% peracetic acid solution. In other embodiments, theweight to weight ratio is about 30:1 Those skilled in the art willrecognize that slight modifications to the concentration can be made toaffect varying degree of oxidation, with concomitant alterations inreaction time, temperature, and liquid to solid ratio. It has also beendiscussed that performic acid offers the advantage of minimal peptidebond cleavage compared to peracetic acid. However, peracetic acid offersthe advantages of cost and availability. In some embodiments, theoxidation temperature is between 0 and 100° Celsius. In a specificembodiment, the oxidation temperature is 37° C. In some embodiments, theoxidation time is between 0.5 and 24 hours. In a specific embodiment,the oxidation time is 12 hours. In some embodiments, mechanical mixingis used to maximize oxidation efficiency. Additional yield can beachieved with subsequent extractions with dilute solutions of oxidant orwater. After oxidation, the keratin protein source can be rinsed free ofresidual oxidant using copious amounts of purified water. In someembodiments, the oxidized keratin protein source is washed with wateruntil residual oxidant is removed. In some embodiments, the washing stepis performed until the washed keratin protein source does not testpositive for oxidant. In a specific embodiment, the washed keratinprotein source has about 5 ppm or less residual oxidant.

The keratoses may be extracted from the oxidized keratin protein sourceusing an aqueous solution of a denaturing agent. Protein denaturants arewell known in the art, including but not limited to, urea, transitionmetal hydroxides (e.g. sodium and potassium hydroxide), ammoniumhydroxide, and tris(hydroxymethyl)aminomethane (Tris, also known asTrizma® base). In some embodiments, Tris is used at a ratio of about 5:1to about 50:1 weight of protein source to a Tris solution of aconcentration of about 0.01 to 1M. In other specific embodiments, theratio is 25:1 or 40:1. In another specific embodiment, Tris is used at aconcentration of 100 mM. Those skilled in the art will recognize thatslight modifications to the concentration can be made to effect varyingdegree of extraction, with concomitant alterations in reaction time,temperature, and liquid to solid ratio. In some embodiments, theextraction temperature is between 0° and 100° Celsius. In a specificembodiment, the extraction temperature is 37° C. In some embodiments,the extraction time is between 0.5 and 24 hours. In a specificembodiment, the extraction time is about 2 hours. Additional yield canbe achieved with subsequent extractions with dilute solutions of Tris orpurified water. Often, the extraction is performed with mechanicalagitation in a mixing tank to ensure a more efficient yield.

Kerateine Production

Similar to the methods described above for extraction and purificationof keratoses, kerateines can be produced by reduction of a keratinprotein source with thioglycolic acid or beta-mercaptoethanol.Specifically, thioglycolic acid (TGA) is often used. In someembodiments, TGA is added to the keratin protein source at a ratio ofabout 5:1 to about 50:1. In a specific embodiment, TGA is added at aratio of 25:1. The TGA is added at a solution ranging in concentrationsfrom about 0.1 to about 10M. In a specific embodiment, the TGA is addedin solution at a concentration of 0.5M. During extraction, mechanicalagitation is used to maximize extraction efficiency.

The solution containing reductant and extracted kerateine proteins(soluble keratin protein solution) is the collected and stored bystraining the keratin protein source through a 400 micron mesh andstoring the solution at 4° C. A base is then added to the drainedkeratin protein source in a ratio of about 10:1 to about 50:1. In aspecific embodiment, the base is added to the drained keratin proteinsource at a ratio of 25:1. In some embodiments, the base is Trisgenerally used at a concentration of about 100 mM. The keratin proteinsource in the solution with base is mixed with agitation of about 2hours at 37° C. The solution containing the base and extracted keratinproteins (soluble keratin protein solution) is then filtered and addedto the first extracted solution and stored.

Those skilled in the art will recognize that slight modifications to theconcentration can be made to effect reduction, with concomitantalterations in pH, reaction time, temperature, and liquid to solidratio. In some embodiments, the reduction is performed at a temperaturebetween 0 and 100° Celsius. In a specific embodiment, the temperature is37° C. In some embodiments, the reduction time is between 0.5 and 24hours. In a specific embodiment, the reduction is performed for 15hours. Unlike the previously described oxidation reaction, reduction iscarried out at basic pH. That being the case, keratins are highlysoluble in the reduction media and are expected to be extracted. Thereduction solution may therefore be combined with the subsequentextraction solutions and processed accordingly. The reduction is carriedout with mechanical agitation in a mix tank to increase the efficiencyof the reduction of the keratin proteins.

Residual reductant and denaturing agents can be removed from solution bydialysis. Typical dialysis conditions are 1 to 2% solution of kerateinesdialyzed against purified water. Those skilled in the art will recognizethat other methods exist for the removal of low molecular weightcontaminants in addition to dialysis (e.g. microfiltration,chromatography, and the like). Once dissolved, the kerateines are stablein solution without the denaturing agent for finite periods. Therefore,the denaturing agent can be removed without the resultant precipitationof kerateines. Regardless of the fractionation/purification process, theresulting kerateines can be concentrated and lyophilized, similar tokeratoses.

A soluble keratin protein solution is produced by the extraction ofkeratose and/or kerateine by either oxidative means for keratose, or byreductive means for kerateine.

In some embodiments, the soluble keratin protein solution may comprise80%, 85%, 90%, 95%, 99% or more keratose. The keratose may bealpha-keratose or gamma-keratose, or some combination thereof. In someembodiments, the keratose in the soluble keratin protein solutioncomprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or morealpha-keratose. In other embodiments, the keratose in the solublekeratin protein solution comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or more gamma-keratose.

In some embodiments, the soluble keratin protein solution may comprise80%, 85%, 90%, 95%, 99% or more kerateine. The kerateine may bealpha-kerateine or gamma kerateine, or some combination thereof. In someembodiments, the kerateine in the soluble keratin protein solutioncomprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or morealpha-kerateine. In other embodiments, the kerateine in the solublekeratin protein solution comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or more gamma-kerateine.

High Speed Centrifugation

In order to remove many of the keratin associated proteins and otherproteins extracted through either oxidative or reductive processeslisted above, a high speed centrifugation step is used. Current methodsknown in the art generally use a low speed centrifugation (around 4,000rpm) to clear particulate matter. However, this speed does not createenough force to remove many of the protein contaminants present in theextracted protein solution. Thus, in some embodiments, high speedcentrifugation is employed. Speeds in excess of about 5,000 rpm to about30,000 rpm can be used. In a specific embodiment, the extracted proteinsolution is spun at about 20,000 rpm to produce a clarified proteinsolution. In another specific embodiment, the high speed centrifugationstep is performed at about 4° C.

A clarified protein solution is produced by the high speedcentrifugation of the soluble keratin protein solution.

In some embodiments, the clarified protein solution may comprise 80%,85%, 90%, 95%, 99% or more keratose. The keratose may be alpha-keratoseor gamma-keratose, or some combination thereof. In some embodiments, thekeratose in the clarified protein solution comprises 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or more alpha-keratose. In otherembodiments, the keratose in the clarified protein solution comprises50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or moregamma-keratose.

In some embodiments, the clarified protein solution may comprise 80%,85%, 90%, 95%, 99% or more kerateine. The kerateine may bealpha-kerateine or gamma kerateine, or some combination thereof. In someembodiments, the kerateine in the clarified protein solution comprises50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or morealpha-kerateine. In other embodiments, the kerateine in the clarifiedprotein solution comprises 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or more gamma-kerateine.

Dialysis

In many instances during protein purification, dialysis is used toseparate or even to concentrate certain protein species present in thesample. Accordingly here, in many embodiments, the clarified proteinsolution is subjected to a dialysis step to fractionate certain proteinspecies. In some embodiments, a 100 kDa molecular weight cutoff membraneis employed in the purification of alpha-keratose or alpha-kerateine. Inother embodiments, a 5 kDa molecular weight cutoff membrane is employedto purify gamma-keratose or gamma kerateine. A common matrix for thedialysis membranes is regenerated cellulose, however, many othermembrane preparations suitable for protein purification may be used.

In many instances, pressure is applied to aid in the dialysis process.If the pressure applied is too low, the resultant solutions containgreater protein fragments and peptides. Conversely, if the pressure istoo high, the result is protein complex degradation. Thus, in someembodiments, the dialysis is performed under conditions that maintain atransmembrane pressure from about 30 psi to about 40 psi (alpha) andabout 50 psi to about 70 psi (gamma). Further, it is important tominimize the heat buildup developed by the shear stress of pressurizeddialysis. Thus, in some embodiments, the dialysis is carried out at atemperature from about 4° C. to about 20° C. In a specific embodiment,the dialysis is carried out at about 15° C. to about 20° C.

Additionally, as the solution is dialyzed, the conductivity is adjusted.In some embodiments, the conductivity is adjusted down to about or below0.6 mS. In some instances, the conductivity is adjusted with water.

Post dialysis, the clarified protein solution may comprise 80%, 85%,90%, 95%, 99% or more keratose. The keratose may be alpha-keratose orgamma-keratose, or some combination thereof. In some embodiments, thekeratose in the clarified protein solution post dialysis comprises 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% ormore alpha-keratose. In other embodiments, the keratose in the clarifiedprotein solution post dialysis comprises 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more gamma-keratose. Inalternative embodiments, the clarified protein solution post dialysis issubstantially free of alpha-keratose. In yet other alternativeembodiment, the clarified protein solution post dialysis issubstantially free of gamma-keratose.

Post dialysis, the clarified protein solution may comprise 80%, 85%,90%, 95%, 99% or more kerateine. The kerateine may be alpha-kerateine orgamma-kerateine, or some combination thereof. In some embodiments, thekerateine in the clarified protein solution post dialysis comprises 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% ormore alpha-kerateine. In other embodiments, the kerateine in theclarified protein solution post dialysis comprises 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or moregamma-kerateine. In alternative embodiments, the clarified proteinsolution post dialysis is substantially free of alpha-kerateine. In yetother alternative embodiment, the clarified protein solution postdialysis is substantially free of gamma-kerateine.

Lyophilization

Storage of proteins for any length of time can pose stability problems.While working with proteins in the lab, they should be kept on ice.Since proteins are generally more stable at colder temperatures,maintenance at low temperatures even for short duration is recommended.Typically, proteins can be freeze-dried (lyophilized) to achieve storageconditions while maintaining protein stability.

In some embodiments, lyophilization is used to produce a protein cake ofpurified protein post-dialysis. The lyophilization is used to stabilizethe extracted keratin proteins. Methods known in the art such as shellfreezing followed by vacuum or bulk freezing and applying high heat tendto degrade proteins. Accordingly, in some embodiments, a keratin proteincake, comprising keratose and/or kerateine is produced by alyophilization of a clarified keratin protein solution, optionally afterdialysis.

In some embodiments, the clarified protein solution post-dialysis isbulk frozen at about −40° C., then a vacuum is applied until thecontainment containing the solution reaches about 250 ton. In someembodiments, heat is then applied in a step-wise fashion, bringing thematerial to about 0° C., then to about 25° C., then to about 37° C.,while maintaining 250 torr pressure. In some embodiments, thelyophilization process occurs over a 24 hour period.

In some embodiments, the keratin protein cake may comprise 80%, 85%,90%, 95%, 99% or more keratose. The keratose may be alpha-keratose orgamma-keratose, or some combination thereof. In some embodiments, thekeratose in the keratin protein cake comprises 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more alpha-keratose.In other embodiments, the keratose in the keratin protein cake comprises30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or more gamma-keratose. In alternative embodiments, the keratinprotein cake is substantially free of alpha-keratose. In yet otheralternative embodiments, the keratin protein cake is substantially freeof gamma-keratose.

In some embodiments, the keratin protein cake may comprise 80%, 85%,90%, 95%, 99% or more kerateine. The kerateine may be alpha-kerateine orgamma-kerateine, or some combination thereof. In some embodiments, thekerateine in the keratin protein cake comprises 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or morealpha-kerateine. In other embodiments, the kerateine in the keratinprotein cake comprises 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or more gamma-kerateine. In alternativeembodiments, the keratin protein cake is substantially free ofalpha-kerateine. In yet other alternative embodiments, the keratinprotein cake is substantially free of gamma-kerateine.

Grinding

Precise grinding of the lyophilized material aids in the homogeneity ofreconstitution and protein stability. Previous methods involve crudegrinding methods, including grinding or chopping of the material in ahousehold blender. In the present invention, some embodiments employ acommercial grinding apparatus to machine the material to a homogenousparticle size. In some embodiments, a pharmaceutical mill is employed.In other embodiments, the particle size is 1 millimeter or less indiameter.

It is also important to remove the static charge from the groundmaterial to make it easier to work with. Accordingly, in someembodiments, the ground material has been deionized.

6. EXAMPLES 6.1 Example 1 Keratose Extraction Methods (OxidativeExtraction)

Untreated Chinese hair was used in the extraction method. The hair wasend-cut to lengths of ¼, ½, ¾ and 1 inch segments and cleaned by washingin a warm water solution.

Step 1: The hair was added to a mixing tank. The tank was a 316Lstainless steel vessel that contained a propeller for mechanicalagitation (see FIG. 1). The oxidant was added to the vessel. The oxidantused was a 2% solution of paracetic acid (PAA) at a 25:1weight-to-weight ratio. The mixture was mechanically mixed for a periodof 12 hours at 37° C. The mechanical mixing resulted in completeoxidation of the hair shafts (see FIG. 2).

Step 2: The residual solution containing the oxidant was drained,neutralized and discarded.

Step 3: The oxidized hair was collected and rinsed with water until PAAtest strips revealed no residual oxidant in the solution.

Step 4: A base was then added to the drained hair in a ratio of 25:1. Inthis Example, a 100 mM Tris base was used. The solution was mixed withmechanical agitation in the mixing tank for 2 hours at 37° C. FIG. 3shows hair that has completed this extraction step.

Step 5: The solution containing base and extracted keratin proteins wasthen collected and stored in a separate container at 4° C. The remaininghair was retained by sieving through a steel mesh with a pore size ofthe mesh of 400 microns. The mechanical agitation employed in this stephelped to remove any residual extracted solution from the remaining hairmass.

Step 6: Purified water was then added to the hair at a ratio of 25:1 andmixed for 2 hours at 37° C.

Step 7: The solution containing water and extracted keratin proteins wasthen collected and added to the solution from Step 5 in a separatecontainer stored at 4° C. In order to get the maximum extraction yield,the hair was sieved through a steel mesh with a pore size of 400 micronsThe mechanical agitation employed ensures removal of any residualextracted solution from the remaining hair mass. FIG. 4 shows the hairshafts after the final water extraction.

Step 8: The combined mixture from Step 5 and Step 7 was then centrifugedat 20,000 rpm to remove any solids or beta-keratins. Centrifugation atspeeds at or below 4,000 rpm does not fully remove residual solids,contributing to poor dialysis and final product.

Step 9: The centrifuged solution was filtered with a 20 micrometer poresize capsule filter.

Step 10: The solution from Step 9 was dialyzed against a 100 kDamolecular weight cut off dialysis membrane, made from regeneratedcellulose, using standard tangential flow filtration. It can bebeneficial to cool the solution to minimize heat from shear forces onpumps. It can also be beneficial to maintain trans-membrane pressuresbetween 30-40 psi during the dialysis process. Lower pressures result insolutions that contain greater protein fragments and peptides, higherpressures result in protein complex degradation. The solution wasdialyzed until the conductivity reached 0.6 mS using additions ofpurified water to replace permeate. The first complete solution wash wascollected and stored in a storage tank at 4° C.

Step 11: The solution from Step 10 was then lyophilized into a keratinprotein cake of alpha keratose. The lyophilization step helps maintainintact keratin proteins. The solution was bulk frozen to −40° C. quicklythen had a vacuum applied until the containment vessel containing theprotein reached 250 torr. Heat was then applied in a step-wise fashionto bring the material first to 0° C., then to 25° C., then to 37° C.while maintaining 250 ton. The temperature was maintained at 37° C. inorder to prevent degradation during the drying process.

Step 12: The first wash solution from Step 10 (containinggamma-keratose) was dialyzed against a 5 kDa molecular weight cut offdialysis membrane made from regenerated cellulose, using standardtangential flow filtration methods. The solution was cooled to minimizethe heat build-up from shear forces on the pumps. Also, thetrans-membrane pressures were maintained between 50-70 psi during thedialysis process. Lower pressures result in solutions that containgreater protein fragments and peptides, higher pressures result inprotein complex degradation. The solution was dialyzed until theconductivity reached 0.6 mS using additions of purified water to replacepermeate.

Step 13: The solution from Step 12 was lyophilized into a keratinprotein cake of gamma-keratose. The solution containing thegamma-keratose was bulk frozen at −40° C. quickly then a vacuum wasapplied until the containment vessel containing the protein reached 250ton. Heat was then applied in a step-wise fashion to bring the materialfirst to 0° C., then to 25° C., then to 37° C. while maintaining 250ton. The mixture was maintained at 4° C. Elevated temperatures in themethod were avoided in order to prevent degradation during the dryingprocess.

Step 14: The keratin protein cakes from Step 11 and 13 wereindependently ground using a pharmaceutical mill with a mesh size of 1millimeter. The ground protein was deionized to better allow furtherprocessing. The ground protein was then placed in sterile bags and isnow ready for use in a variety of medical and research applications.

6.2 Example 2 Kerateine Extraction Methods (Reductive Extraction)

Kerateine Extraction Methods (Reductive Extraction).

Untreated Chinese hair was end-cut to lengths of ¼, ½, ¾ and 1 inchsegments and washed in a warm water solution.

Step 1: The hair was added to a 316L stainless steel vessel thatcontained a propeller for mechanical agitation (see FIG. 1). Thereductant was added to the vessel. The reductant was a 0.5M solution ofa thioglycolic acid (TGA) at a ratio of 25:1. The mixture wasmechanically mixed for a period of 15 hours at 37° C. The mechanicalmixing can be beneficial to improve the extent to which reduction occursin the hair shafts.

Step 2: The solution containing reductant and extracted keratin proteinswas collected and stored in a separate container at 4° C. The remaininghair was retained by sieving through a steel mesh with a pore size of400 microns. The mechanical agitation applied during the strainingprocess helps to collect as much solution as possible from the hairmass.

Step 3: A base was then added to the drained hair in a ratio of 25:1.The base used here was a 100 mM Tris base solution. The solution wasmixed with mechanical agitation in a mixing tank for 2 hours at 37° C.

Step 4: The solution containing base and extracted keratin proteins wascollected and added to the solution from Step 2 and stored at 4° C. Theremaining hair was retained by sieving through a steel mesh with a poresize of 400 microns. The mechanical agitation applied during thestraining process helps to collect as much solution from the hair mass.

Step 5: Purified water was added to the hair at a ratio of 25:1 andmixed for 2 hours at 37° C.

Step 6: The solution containing water and extracted keratin proteins wascollected and added to the solution from Step 4 and stored at 4° C. Inorder to maximize extraction yield, the hair was sieved through a steelmesh with a pore size of 400 microns. Mechanical agitation was appliedduring the straining process to strain as much solution as possible fromthe hair mass.

Step 7: A second reduction step was needed to fully extract the keratinproteins from the hair shaft. The reductant used was a 0.5M solution ofa thioglycolic acid (TGA) at a ratio of 25:1. The mixture wasmechanically mixed for a period of 15 hours at 37° C. Mechanical mixingwas used to ensure complete reduction of the hair shafts.

Step 8: The solution containing reductant and extracted keratin proteinswas collected and stored in a separate vessel containing the solutionfrom Step 6 and stored at 4° C. The remaining hair was retained bysieving through a steel mesh with a pore size of 400 microns. Mechanicalagitation applied during the straining process helps to strain as muchsolution from the hair mass.

Step 9: A base was added to the drained hair in a ratio of 25:1. Thebase used was a 100 mM Tris base solution. The solution was mixed withmechanical agitation in a mixing tank for 2 hours at 37° C.

Step 10: The solution containing base and extracted keratin proteins wasthen collected and added to the solution from Step 8 and stored at 4° C.The remaining hair was retained by sieving through a steel mesh with apore size of 400 microns. Mechanical agitation applied during thestraining process helps to strain as much solution as possible from thehair mass.

Step 11: Purified water was then added to the hair at a ratio of 25:1and mixed for 2 hours at 37° C.

Step 12: The solution containing water and extracted keratin proteinswas collected and added to the solution from Step 10 stored at 4° C. Inorder to get the maximum extraction yield, the hair was sieved through asteel mesh of a pore size of 400 microns. Again, mechanical agitationapplied during the straining process helps to strain as much solution aspossible from the hair mass.

Step 13: The combined mixture from Steps 12, 10, 8, 6, 4, and 2 wascentrifuged at 20,000 rpm to remove any solids or beta keratins.Centrifugation at speeds at or below 4,000 rpm does not fully removeresidual solids, contributing to poor dialysis and final product.

Step 14: The centrifuged solution was filtered with a 20 micrometer poresize capsule filter.

Step 15: The solution from Step 14 was dialyzed against a 100 kDamolecular weight cut off regenerated cellulose dialysis membrane usingstandard tangential flow filtration methods. The solution was cooled todissipate the heat from shear forces on pumps. Also, trans-membranepressures were maintained between 30-40 psi during the dialysis process.The solution was dialyzed until the conductivity lowered from 24 mS to0.6 mS using additions of purified water to replace permeate. Thisrequired about 5 complete volume changes (or washes) and left someresidual TGA in the solution. The TGA can be completely removed bydialyzing until the conductivity reaches 0 mS or 12-20 volume changes.The first complete solution wash was collected and stored in a storagetank at 4° C.

Step 16: The solution from Step 15 was lyophilized into a keratinprotein cake of alpha keratin. A lyophilization step was used tomaintain intact keratin proteins. Generally, methods that are known inthe art such as shell freezing followed by vacuum or bulk freezing andapplying high heat tend to degrade the proteins. Here, thelyophilization step was to bulk freeze the solution to −40° C. quicklythen apply a vacuum until the containment vessel containing the proteinreached 250 ton. Heat was applied in a step-wise fashion to bring thematerial first to 0° C., then to 25° C., then to 37° C. whilemaintaining 250 torr. The temperature was not allowed to exceed 37° C.in order to prevent degradation during the drying process.

Step 17: The first wash solution from Step 15 (gamma kerateine) wasdialyzed against a 5 kDa molecular weight cut off regenerated cellulosedialysis membrane using standard tangential flow filtration methods.Heat from shear forces on pumps was minimized by cooling the solution.Also, trans-membrane pressures between 50-70 psi were maintained duringthe dialysis process. Lower pressures result in solutions that containgreater protein fragments and peptides, higher pressures result inprotein complex degradation. The solution was dialyzed until theconductivity reached 0.6 mS using additions of purified water to replacepermeate.

Step 18: The solution from Step 17 was lyophilized into a keratinprotein cake of gamma-kerateine. Here, lyophilization was accomplishedby bulk freezing the solution to −40° C. quickly then applying a vacuumuntil the containment vessel containing the protein reached 250 torr.Heat was then applied in a step-wise fashion to bring the material firstto 0° C., then to 25° C., then to 37° C. while maintaining 250 ton. Thetemperature was not allowed to exceed 37° C. in order to preventdegradation during the drying process.

Step 19: The keratin protein cakes from Step 18 and 16 wereindependently ground using a pharmaceutical mill with a mesh size of 1millimeter. The ground protein was deionized to better allow furtherprocessing. The ground protein was then placed in sterile bags and isnow ready for use in a variety of medical and research applications.

What is claimed is:
 1. A method for extracting keratin proteinscomprising: a. treating a keratin protein source with an oxidizing orreducing agent to solubilize keratin proteins; b. separating the solubleproteins from the keratin protein source by high speed centrifugation toproduce a clarified soluble keratin protein solution; and c.lyophilizing the clarified soluble keratin protein solution into akeratin protein cake.
 2. The method of claim 1, wherein said keratinprotein source is selected from the group consisting of hair, wool, fur,horns, hooves, beaks, feathers, and scales.
 3. The method of claim 1,wherein said high speed centrifugation is performed at 5,000 rpm orhigher.
 4. (canceled)
 5. The method of claim 1, wherein said methodcomprises a dialysis step. 6.-8. (canceled)
 9. The method of claim 5,wherein said clarified protein solution is substantially free of proteinfragments or peptides after dialysis. 10.-11. (canceled)
 12. The methodof claim 1, wherein said method comprises mechanical agitation. 13.-17.(canceled)
 18. The method of claim 1, wherein said method comprises anoxidizing reagent.
 19. The method of claim 18, wherein said oxidizingreagent comprises peracetic acid.
 20. The method of claim 18 whereinsaid clarified protein solution is substantially free of the oxidizingreagent. 21.-25. (canceled)
 26. The method of claim 1, wherein saidkeratin protein is keratose.
 27. The method of claim 1, wherein saidmethod comprises a reducing agent.
 28. (canceled)
 29. The method ofclaim 27, wherein said clarified protein solution is substantially freeof the reducing agent.
 30. The method of claim 27, wherein saidclarified protein solution comprises at least 90% or more kerateine.31.-34. (canceled)
 35. The method of claim 1, wherein said keratinprotein is kerateine.
 36. A method for extracting keratin proteinscomprising: a. treating a keratin protein source with an oxidizing orreducing agent to solubilize keratin proteins; b. separating the solubleproteins from the keratin protein source by high speed centrifugation toproduce a clarified soluble keratin protein solution; and c. dialyzingthe clarified soluble keratin protein solution to obtain a solublekeratin protein solution which exhibits a conductivity of 0.6 mS orless.
 37. The method of claim 36, wherein said high speed centrifugationis performed at 5,000 rpm or higher. 38.-39. (canceled)
 40. The methodof claim 36, wherein said dialysis is performed at a pressure from about10 psi to about 70 psi.
 41. The method of claim 40, wherein the dialysispermeate comprises gamma-keratose. 42.-44. (canceled)
 45. The method ofclaim 36, wherein said keratin protein is keratose. 46.-47. (canceled)48. The method of claim 36, wherein said keratin protein is kerateine.